1. Field of the Invention
The present invention includes a method for combining immunostaining and FISH using covalently binding small molecule tags.
2. Description of the Related Art
Many methods are known to aid in the microscopic analysis of samples. For example, without limitation, it is known that certain dyes have an affinity for certain cellular structures. Such dyes may therefore be used to aid in analysis by helping to further elucidate such structures.
Fluorescence microscopy of cells and tissues is well known in the art. Methods have been developed to image fluorescent cells in a microscope and extract information about the spatial distribution and temporal changes occurring in these cells. Some of these methods and their applications are described in an article by Taylor, et al. in American Scientist 80 (1992), p. 322-335. These methods have been designed and optimized for the preparation of a few specimens for higher spatial and temporal resolution imaging measurements of distribution, amount and biochemical environment of the fluorescent reporter molecules in the cells.
Detection of fluorescent signals may be by way of an epifluorescent microscope which uses emitted fluorescent light to form an image (whereas a conventional reflecting microscope uses scattered illumination light to form an image). The excitation light of an epifluorescence microscope is used to excite a fluorescent tag in the sample causing the fluorescent tag to emit fluorescent light. The advantage of an epifluorescence microscope is that the sample may be prepared such that the fluorescent molecules are preferentially attached to the biological structures of interest thereby allowing identification of such biological structures of interest.
The acronym “FISH” references a technique that uses chromophore tags (fluorophone) that emists a secondary signal if illuminated with an light to detect a chromosomal structure. FISH uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity. Such tags may be directed to specific chromosomes and specific chromosome regions. The probe has to be long enough to hybridize specifically to its target (and not to similar sequences in the genome), but not too large to impede the hybridization process. Typically, the probe is tagged directly with fluorophores. This can be done in various ways, for example nick translation or PCR using tagged nucleotides. If signal amplification is necessary to exceed the detection threshold of the microscope (which depends on many factors such as probe labelling efficiency, the kind of probe and the fluorescent dye), secondary antibodies or streptavidin are bound to the tag molecules, thus amplifying the signal.
The FISH technique may be used for identifying chromosomal abnormalities and gene mapping. For example, a FISH probe to chromosome 21 permits one to “fish” for cells with trisomy 21, an extra chromosome 21, the cause of Down syndrome. FISH kits comprising multicolor DNA probes are commercially available For example. AneuVysion Multicolor DNA Probe Kit sold by the Vysis division of Abbott Laboratories, is designed for in vitro diagnostic testing for abnormalities of chromosomes 13, 18, 21, X and Y in amniotic fluid samples via fluorescence in situ hybridization (FISH) in metaphase and interphase nuclei. The AneuVysion Assay (CEP 18, X, Y-alpha satellite, LSI 13 and 21) Multi-color Probe Panel uses CEP 18/X/Y probe to detect alpha satellite sequences in the centromere regions of chromosomes 18, X and Y and LSI 13/21 probe to detect the 13q14 region and the 21q22.13 to 21q22.2 region. The combination of colors evidenced is used to determine whether there is normal chromosome numbers or trisomy. In a similar vein, the UroVysion kit by the Vysis division of Abbott Laboratories is designed to detect chromosomal abnormalities associated with the development and progression of bladder cancer by detecting aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus via fluorescence in situ hybridization (FISH) in urine specimens from persons with hematuria suspected of having bladder cancer.
Another process for detecting structures of interest is immunostaining. Immunostaining refers to the laboratory process of detecting biological materials using antibodies. Often these antibodies are labeled with a fluorescent compound which can be viewed by a microscope. Antibodies that detect, for example, a protein of interest in the biological sample are generated by a foreign host species (a polyclonal antibody) or cultured immune cell clones (monoclonal antibodies). After exposure to the foreign protein, the antibodies can be harvested and used as very specific and sensitive detection agents. Antibodies so generated are known as “primary antibodies,” as they bind directly to the protein of interest. Certain immunostaining agents can be applied in a single stage, where the primary antibody is directly linked to a colouring agent. In other cases, the primary antibody is targeted by a “secondary” antibody, targeting a species-specific part of the structure of the primary antibody. The later technique may be advantageous in that the signal is amplified, as multiple secondary antibodies will bind to a primary antibody. It also allows for a high variety of primary antibodies—researchers can make their own antibodies and not have to conjugate them to a colouring agent themselves. Finally, it means that a variety of colouring agents can be conjugated to any given species of secondary antibody, and are available in ready supply. This has opened the door to “double-labelling” experiments, where several proteins can be co-localised.
Traditionally the combination of immunostaining techniques with FISH has been challenging. If immunostaining is performed first, subsequent FISH treatment may abolish mostly non-covalent antibody-antigen interaction. Similarly, if FISH is performed first, the subsequent antibody treatment may release the FISH probe because of its low salt concentration. As the immunostaining of certain portions of a biological sample as well as the FISH staining of other areas of the same biological sample may be advantageous, it would be advantageous if a system allowing for combined immunostaining and FISH staining could be developed.